Column chromatography is a commonly used technique for separation and purification of particular types of molecules from complex sample solutions and complex sample mixtures that include solutes and suspended or partially solvated chemical entities, such as membrane fragments. A chromatography column is prepared by suspending a resin in a buffer solution to form a resin slurry, and then packing the resin slurry within a chromatography tube to form a matrix within the chromatography tube by following a packing procedure, or packing mode. The matrix constitutes the solid phase or stationary phase within the chromatography column. A complex solution that contains one or more types of molecules to be purified, each type referred to as a “target molecule,” is loaded onto the chromatography column in which buffer conditions are established to promote separation of the one or more target molecules from the complex solution. A buffer solution, mobile phase, or eluant solution is then directed through the chromatography column to move desired target molecules and undesired sample-solution components through the chromatography column. Different types of solutes move through the chromatography column at different rates, depending on their different mobilities in, and different affinities for, the mobile phase and the stationary phase, resulting in separation of the one or more target molecules from solutes and suspended entities present in the original sample solution. Solutions containing the one or more target molecules, referred to as “eluates,” are subsequently eluted from the chromatography column.
A chromatography column is generally incorporated within a chromatography system that includes one or more pumps, eluate collectors, and detectors. Column chromatography systems are frequently used for purifying biomolecules, including proteins and other biopolymers, from complex solutions and mixtures, such as, for example, purifying recombinant proteins from cell lysates and cell filtrates. After a target molecule has been partially purified by an initial column-chromatography step and collected in an eluate pool, the partially purified target molecule is often subjected to further purification steps, generally referred to as “polishing steps.”
Although column chromatography is commonly used to purify various target molecules, certain problems are frequently encountered. It can be difficult to efficiently prepare an eluate containing a target molecule for subsequent polishing steps. In certain cases, the eluate contains a high, residual salt concentration that makes the eluate unsuitable for a next polishing step. In these cases, a buffer may be added to dilute the eluate, resulting in an increased the cost and slowing of the purification process. Researchers, pharmaceutical manufacturers, chromatography-column and matrix manufacturers and vendors, and users of chromatography-based purification methods have recognized the need for improved polishing steps for use in multi-step purification processes.